1. Notes

2. Result Statistics

Figure 1. False discovery rate (FDR) curve. X axis is the number of peptide-spectrum matches (PSM) being kept. Y axis is the corresponding FDR.

Figure 2. PSM score distribution. (a) Distribution of PEAKS peptide score; (b) Scatterplot of PEAKS peptide score versus precursor mass error.

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(b)

Figure 3. De novo result validation. Distribution of residue local confidence: (a) Residues in de novo sequences validated by confident database peptide assignment; (b) Residues in "de novo only" sequences.

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(b)

Table 1. Statistics of data. # of MS scans 8306 # of MS/MS scans 166067 Table 2. Result filtration parameters. Peptide -10lgP ≥15 Peptide Ascore ≥0 Protein -10lgP ≥20 Proteins unique peptides ≥2 De novo ALC Score ≥50% Table 3. Statistics of filtered result. Peptide-Spectrum Matches 27102 Peptide sequences 18699 Protein groups 1436 Proteins 1438 Proteins (#Unique Peptides) 1139 (>2); 299 (=2); 0 (=1); FDR (Peptide-Spectrum Matches) 24.8% FDR (Peptide Sequences) 32.2% FDR (Protein) 36.2% De Novo Only Spectra 23895

Table 4. PTM profile. Name ∆Mass Position #PSM -10lgP Area AScore Oxidation 15.99 M 4651 167.07 4.21E5 1000.00 Carbamidomethyl 57.02 C 1690 160.78 4.37E4 1000.00 Deamidation .98 NQ 1657 159.17 4.86E5 30.46 Phosphorylation 79.97 STY 706 98.88 2.1E5 7.77 Methylation(KR) 14.02 KR 592 91.35 1.39E5 129.76 Acetylation 42.01 N-term 422 80.14 6.63E5 1000.00 Acetylation 42.01 K 335 63.16 6.32E5 7.77 Dehydration -18.01 DSTY 334 75.49   0.00 Sodium 21.98 DE,C-term 216 44.87 7.11E4 34.81 Acetylation 42.01 Protein N-term 202 128.08 2.49E4 1000.00 Carbamylation 43.01 K,N-term 190 64.50   67.18 Dihydroxy 31.99 FKPRWY 185 63.64   51.59 Formylation 27.99 K,N-term 168 64.76   0.00 Didehydro -2.02 STY,C-term 140 75.26 2.62E5 11.06 Amidation -.98 C-term 93 51.55   1000.00 Dimethylation(KR) 28.03 KR 92 107.54 4.37E5 158.52 Carboxylation 43.99 DKW 86 75.19 2.61E4 1000.00 Biotin 226.08 K,N-term 79 33.98 3.93E5 9.09 Sulfation 79.96 STY 61 36.06 4.9E4 45.16 Lysaminoadipicsealde -1.03 K 39 56.64 3.61E4 126.75 Hexose 162.05 NSY 37 36.37 2.68E5 32.21 Methylation(others) 14.02 DEHST 37 78.78   11.10 Deamidation .98 R 37 71.56 2.01E5 1000.00 Pyro-glu from Q -17.03 N-term 36 77.72   1000.00 Carboxylation 43.99 E 26 33.74 3.72E4 25.70 Propionamide 71.04 K,N-term 25 77.03 1.98E4 189.50 Ammonium 17.03 DE,C-term 22 66.25   33.81 Oxidation 15.99 HW 20 54.01 6.52E4 1000.00 Guanidination 42.02 K 18 152.42 3.16E5 5.81 Amidine 41.03 K,N-term 17 40.03 1.17E5 90.01 Cation:Li 6.01 DE 15 54.63 3.75E4 20.80 Ubiquitin 114.04 KS 14 72.65 2.24E5 43.86 Cation:Ca[II] 37.95 DE,C-term 12 59.22 9.72E3 12.42 Sulphone 31.99 M 12 60.35 2.5E5 1000.00 Cation:K 37.96 DE 12 108.45 2.77E4 0.00 Propargylamine 37.03 DE,C-term 11 43.87 1.95E5 159.65 HexNAc 203.08 N 10 30.61   27.94 Met->Aha -4.99 M 10 55.12 9E4 0.00 MethylamineST 13.03 ST 9 56.11 1.3E5 0.00 Methyl+Deamidated 15.00 NQ 8 48.61   1000.00 Ammonia loss -17.03 N 8 48.21 6.16E4 1000.00 Pyro-glu from E -18.01 N-term 8 22.79   1000.00 Acetyl 42.01 SY 7 26.00 4.13E4 32.52 Im_biotin 225.09 N-term 7 38.53 3.93E4 78.74 SeMet 47.94 M 7 40.24 2.82E5 1000.00 EtOH 44.03 KR 7 20.17 2.43E5 1000.00 HNE 156.12 K 6 55.09 1.53E5 1000.00 azole -20.03 S 6 22.57 3.33E5 1000.00 DSP 88.00 K,N-term 6 37.92 7.28E4 61.82 Phosphorylation 79.97 DR 5 19.11 1.76E6 22.45 Carboxymethyl 58.01 K,N-term 5 28.03 6.76E3 52.32 Deoxy -15.99 ST 5 21.38 2.22E5 0.00 Lipoyl 188.03 K 5 70.36   9.75 ISD_z+2_ion -15.01 N-term 5 23.57   1000.00 Ethyl 28.03 D,N-term 5 25.15 9.83E4 0.00 O-Diisopropylphosphate 164.06 K,N-term 5 39.89 3.17E4 1000.00 Oxidation 15.99 DKPR 4 22.05 1.3E6 0.00 ethylamino 27.05 S 4 26.87 8.81E4 34.08 Dihydroxyimidazolidine 72.02 R 4 26.11 7.12E4 1000.00 Pyridylacetyl 119.04 K,N-term 4 34.10   67.12 Aminoadipic 14.96 K 4 40.94 8.69E4 12.35 Maleimide 97.02 K 4 21.88 8.61E4 1000.00 Aminotyrosine 15.01 Y 4 39.52 2.69E3 30.64

3. Experiment Control

Figure 4. Precursor mass error of peptide-spectrum matches (PSM) in filtered result. (a) Distribution of precursor mass error in ppm; (b) Scatterplot of precursor m/z versus precursor mass error in ppm.

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Table 5. Number of identified peptides in each sample by the number of missed cleavages

Missed Cleavages 0 1 2 3 4+ Virulento 12417 4980 1082 220 0

4. Other Information

Table 6. Search parameters. Search Engine Name: PEAKS Parent Mass Error Tolerance: 0.07 Da Fragment Mass Error Tolerance: 0.07 Da Precursor Mass Search Type: monoisotopic Enzyme: Trypsin Max Missed Cleavages: 3 Non-specific Cleavage: none Fixed Modifications:   Carbamidomethylation: 57.02 Variable Modifications:   Oxidation (M): 15.99   Acetylation (K): 42.01   Acetylation (Protein N-term): 42.01   Acetylation (N-term): 42.01   Amidation: -0.98   Beta-methylthiolation: 45.99   Biotinylation: 226.08   Carbamylation: 43.01   and 304 more... Max Variable PTM Per Peptide: 3 Database: Paracoccidioides_Pb18 Taxon: All Searched Entry: 7000 FDR Estimation: Enabled De novo score (ALC%) threshold: 15 Peptide hit threshold (-10logP): 30.0 Peaks run ID: 12 Different data refine parameters are used for this search:

Table 7. Instrument parameters. Fractions: V_PB18_1_RA5_01_1474.d, V_PB18_2_RA6_01_1475.d, V_PB 18_3_RA7_01_1476.d Ion Source: ESI(nano-spray) Fragmentation Mode: CID, CAD(y and b ions) MS Scan Mode: Quadrupole MS/MS Scan Mode: Time of Flight (TOF)